Proteins having a non-naturally occurring amino acid integrated thereinto (hereinafter referred to as “alloproteins”), in which an amino acid residue at a desired location in a protein is substituted with an amino acid other than 20 kinds of amino acids normally involved in protein synthesis (non-naturally occurring amino acids), offer effective means for the functional or structural analysis of proteins. For example, proteins containing a non-naturally occurring amino acid are used for intramolecular labeling, crosslinking of proteins and structural analysis by X-ray or NMR (see, for example, Non-Patent Document 1) and analysis of a signal transduction system (see, for example, Non-Patent Document 2). In order to efficiently produce an alloprotein having a non-naturally occurring amino acid specifically introduced thereinto, it is inevitable to expand a genetic code system by modifying specificity of an aminoacyl-tRNA synthetase (hereinafter referred to as “aaRS”) to tRNA or an amino acid.
As an expression method of such an alloprotein, a method for introducing a phenylalaninyl-tRNA synthetase•tRNAPhe pair from budding yeast into E. coli, thereby amber codon-specifically introducing p-fluorophenylalanine was first reported (see Non-Patent Document 3). At present, the expansion of a genetic code is successfully achieved in E. coli which is a eubacteria and in a eucaryote (see Non-Patent Document 4 regarding a wheat germ extract; and Non-Patent Document 5 regarding a mammalian cell). In all of these examples, a pair of a TyrRS mutant and an amber-suppressor tRNATyr is introduced. However, though a eubacteria type TyrRS•tRNATyr and an archaebacterium/eucaryote type TyrRS•tRNATyr are aminoacylated within each of the groups, it is the key that they are in an orthogonal relation that they cannot be aminoacylated between the groups each other. For example, since a TyrRS•tRNATyr pair of an archaebacterium Methanococcus jannaschii becomes an orthogonal pair in an E. coli system, whereas a pair of E. coli TyrRS and Bacillus stearothermophilus tRNATyr becomes an orthogonal pair in a mammalian cell system, they are used for the expansion of artificial genetic codes thereof (see, for example, Patent Document 1 and Non-Patent Document 5).
On the other hand, phosphoserine is one of amino acids playing a very important role for signal transduction or the like in living bodies. Phosphoserine is in general produced upon phosphorylation of a serine residue in a protein by a specific protein kinase within a mammalian cell. However, there has been no precedent in which this amino acid is successfully site-specifically introduced into a protein viatranslation during protein synthesis. A chief reason for this resides in the matter that it is difficult to design a modified aaRS capable of recognizing phosphoserine. However, in recent years, it has been reported that methanogenic archaebacteria have a phosphoseryl-tRNA synthetase which is an aaRS capable of recognizing phosphoserine (see, for example, Non-Patent Document 6). According to this, various methanogenic archaebacteria lack a cysteinyl-tRNA synthetase (CysRS), and instead of this, a synthesis route of Cys-tRNACys by a two-step reaction in which tRNACys is acylated with a phosphoseryl-tRNA synthetase (SepRS), and the produced phosphoseryl (Sep)-tRNACys is converted into Cys-tRNACys with a Sep-tRNA:Cys-tRNA synthetase is elucidated. All of the documents cited in this specification are incorporated herein by reference.
[Patent Document 1] WO 2004/070024
[Non-Patent Document 1] Hendrickson, W. A., et al., The EMBO Journal, 1990, Vol. 9, pp. 1665-72
[Non-Patent Document 2] Nowak M. W., et al., Science, 1995, Vol. 268, pp. 439-42
[Non-Patent Document 3] Furter, R., Protein Science, 1998, Vol. 7, pp. 419-26
[Non-Patent Document 4] Kiga, D., et al., Proc Natl Acad Sci USA, 2002, Vol. 99, pp. 9715-20
[Non-Patent Document 5] Sakamoto, K., et al., Nucleic Acids Research, 2002, Vol. 30, pp. 4692-4699
[Non-Patent Document 6] Sauerwald, A., et al., Science, 2005, Vol. 307, pp. 1969-1972
The entire disclosures of Patent Document 1 and Non-Patent Documents 1 to 6 are incorporated herein by reference thereto. The following analyses are given by the present invention.